Development of fetal hemoglobin inducers targeting epigenetic and oxidative stress mechanisms

项目总结 β-Hb病是世界上最常见的遗传性血液疾病，但疗效有限 在过去的三十年里，人们已经开发出了治疗方法。诱导正常，但发育迟缓 胎儿血红蛋白(HBF)的表达可减少贫血，改善镰状细胞病(SCD)的临床严重程度。 组蛋白脱乙酰酶(HDAC)被证明能促进成人胎儿γ-珠蛋白基因的沉默 红系细胞和HDAC抑制剂被证明能增加β-血红蛋白病患者的HBF。但 由于静脉给药的需要和它们的抗病毒作用，这些药物的应用受到了限制。 增殖效应。本项目致力于开发安全有效的小分子口服HBF诱导剂。 在一项研究中，我们观察到一种新型的丁酸(BA)和δ-氨基酮戊酸酯的口服结合物诱导HbF。 (ALA)，命名为AN-233，以及BACH1抑制剂，促进镰状红系祖细胞NRF2的表达 和β-yac小鼠。接下来，聚焦于这些小分子制剂，我们将检验中心假设 表观遗传组蛋白修饰和增强核转录因子2结合对γ-珠蛋白基因转录的调控 介导HBF诱导。具体目的1.将检验口服前药an-233诱发HBF的预测 通过表观遗传组蛋白修饰和NRF2激活表达，使用相关的临床前 动物模型。在β-yac和scd转基因小鼠上进行的治疗将确定最佳口服剂量 和AN233的时间表，诱导HBF而不产生抗增殖作用。的表型效应 AN-233在SCD小鼠中与氧化应激有关，并将检测其抗镰刀作用的能力。要达到一个指标 在人类药物疗效方面，我们将测试AN-233在贫血幼崽中的作用，以确定HBF的诱导和效果 关于造血术。我们将通过涉及表观遗传组蛋白的AN-233来确定γ-珠蛋白的激活机制 乙酰化以及ALA在血红素合成和NRF2激活中的作用。具体目标2.检验预测 口服BACH1抑制剂HPP-D增强NRF2与γ-珠蛋白启动子的结合将被激活 抄写。我们将建立BACH1抑制剂HPP-D，作为使用镰状红系祖细胞的HBF诱导剂 并与临床相关药物进行联合治疗，包括羟基脲和地西他滨。 随后，HPP-D通过增强γ中NRF2的结合而诱导HbF的分子机制被证实。 将对珠蛋白启动子进行评估。最后，HPP-D在临床前SCD中诱导HBF表达的能力 小鼠将在体内建立疗效。预期结果包括建立特定的有效的HDAC抑制剂和 调节β-珠蛋白基因座染色质结构以利于γ-珠蛋白转录的药物，用于后续临床 针对SCD改进疾病修正疗法的未满足需求的研究。

PROJECT SUMMARY The β-hemoglobinopathies are the most common genetic blood disorders worldwide, but limited effective treatments have been developed over the last three decades. Induction of normal, but developmentally silenced fetal hemoglobin (HbF) expression reduces anemia and ameliorates clinical severity of sickle cell disease (SCD). Histone deacetylases (HDACs) have been shown to promote silencing of the fetal γ-globin genes in adult erythroid cells, and HDAC inhibitors were shown to increase HbF in patients with β-hemoglobinopathies. But these had limitations for pharmaceutical application due to the need for intravenous administration and their anti- proliferative effects. This project focuses on creating safe and effective small-molecule oral HbF-inducing agents. In one study, we observed HbF induction with a novel oral conjugate of butyric acid (BA) and δ-aminolevulinate (ALA), named AN-233, and a BACH1 inhibitor that enhanced NRF2 expression in sickle erythroid progenitors and β-YAC mice. Next, focusing on these small-molecule agents, we will test the central hypothesis that modulation of γ-globin gene transcription through epigenetic histone modifications and enhanced NRF2 binding mediate HbF induction. Specific Aim 1. will test the prediction that the oral prodrug AN-233 induces HbF expression through epigenetic histone modifications and NRF2 activation, using relevant preclinical animal models. Treatments conducted in β-YAC and SCD transgenic mice will establish the optimal oral dose and schedule of AN233 that induces HbF without producing anti-proliferative effects. The phenotypic effects of AN-233 in SCD mice related to oxidative stress, and its anti-sickling ability will be tested. To achieve an indicator of human drug efficacy we will test AN-233 in anemic juvenile baboon to determine HbF induction and effects on hematopoiesis. We will define mechanisms of γ-globin activation by AN-233 involving epigenetic histone acetylation and the role of ALA in heme synthesis and NRF2 activation. Specific Aim 2. Test the prediction that the oral BACH1 inhibitor HPP-D enhances NRF2 binding to the γ-globin promoter ARE to activate transcription. We will establish the BACH1 inhibitor HPP-D, as an HbF inducer using sickle erythroid progenitors and conduct combination drug treatments with clinically relevant agents including hydroxyurea and decitabine. Subsequently, molecular mechanisms of HbF induction by HPP-D through enhanced NRF2 binding in the γ- globin promoter will be evaluated. Finally, the ability of HPP-D to induce HbF expression in the preclinical SCD mice will establish in vivo efficacy. Expected outcomes include establishing specific potent HDAC inhibitors and agents that modulate β-globin locus chromatin structure in favor of γ‐globin transcription, for subsequent clinical studies to address the unmet need for improved disease-modifying therapy for SCD.